These authors contributed equally to this work.
Article first published online: 23 FEB 2012
Copyright © 2011 American Association for the Study of Liver Diseases
Volume 55, Issue 3, pages 730–741, March 2012
How to Cite
Wang, S., Qiu, L., Yan, X., Jin, W., Wang, Y., Chen, L., Wu, E., Ye, X., Gao, G. F., Wang, F., Chen, Y., Duan, Z. and Meng, S. (2012), Loss of microRNA 122 expression in patients with hepatitis B enhances hepatitis B virus replication through cyclin G1-modulated P53 activity. Hepatology, 55: 730–741. doi: 10.1002/hep.24809
Potential conflict of interest: Nothing to report.
Supported by a grant from Major State Basic Research Development Program of China (No. 2007CB512802), grants from the National Natural Science Foundation of China (NSFC, 81021003, 91029724), grants from Key Projects in the National Science & Technology Program of Eleventh 5-year Plan (2008ZX10002-005-3, 2008ZX10002-009), and the CAS projects (KSCX2-YW-R-1, KSCX2-YW-R-183, 2010-Biols-CAS-0204).
- Issue published online: 23 FEB 2012
- Article first published online: 23 FEB 2012
- Accepted manuscript online: 22 NOV 2011 06:30AM EST
- Manuscript Accepted: 4 NOV 2011
- Manuscript Received: 19 DEC 2010
Additional Supporting Information may be found in the online version of this article.
|HEP_24723_sm_suppinfotable1.doc||25K||Supporting Information Table 1. Correlation between miR-122 and cyclin G1 status in patients|
|HEP_24723_sm_suppinfofig1.tif||21K||Supporting Information Fig. S1. HepG2 cells were co-transfected with 50 nM of the miR-122 expression plasmid pSuper-122 or the empty plasmid pSuper as mock and pHBV. The levels of HBsAg and HBeAg in the supernatant were detected by ELISA at 72h after transfection. The data are presented as the mean ± SD from three independent experiments. **p < 0.01 compared with mock.|
|HEP_24723_sm_suppinfofig2.tif||105K||Supporting Information Fig. S2. Cyclin G1 expression in transfected HepG2 cells. (A) Epifluorescent micrograph of EGFP expression in EGFP-G1 transfected cells. EGFP expression was assessed by fluorescence microscopy at 24 hr after plasmid delivery. The experiments were performed twice and similar results were obtained. (B) Real-time PCR and Western blot analyses of cyclin G1 expression in cyclin G1 siRNA transfected cells. Error bars, the mean ± S.D. of three independent experiments. ** p <0.01 compared with mock. The experiments were performed twice, and similar results were obtained.|
|HEP_24723_sm_suppinfofig3.tif||120K||Supporting Information Fig. S3. HepG2 cells were co-transfected with pHBV and cyclinG1 siRNA (si-cyclin G1) or a scrambled siRNA as mock. The levels of HBsAg and HBeAg in the supernatant were detected by ELISA (A). HBV-DNA levels were detected by real-time PCR (B), and HBV RNAs were determined by real-time PCR and Northern blot (C). Data are presented as the mean ± SD from three independent experiments. **p < 0.01 compared with mock.|
|HEP_24723_sm_suppinfofig4.tif||1740K||Supporting Information Fig. S4. Photomicrographs of cyclin G1 expression on immunohistochemistry. Paraffine tissue sections from CHB (chronic hepatitis B), CSHB (chronic severe hepatitis B) and healthy controls were stained with anti-cyclin G1 antibody and the HRP-conjugated secondary antibody, using DAB for visualization.|
|HEP_24723_sm_suppinfofig5.tif||26K||Supporting Information Fig. S5. Western blot analysis of p53 levels in pcDNA-p53 or mock (the empty vector) transfected HepG2 cells. The protein levels of p53 were measured by immunoblot 72h post-transfection. GAPDH was used as a loading control. The ratios of the band intensity were shown. The experiments were performed twice, and similar results were obtained.|
|HEP_24723_sm_suppinfofig6.tif||107K||Supporting Information Fig. S6. p53 supresses HBV replication. HepG2 cells were co-tranfected with pcDNA-p53 or mock (the empty vector) and pHBV. The secretion of HBsAg and HBeAg was measured by ELISA (A). HBV-DNA levels in cells were detected by Southern blot (B). rcDNA, relaxed circular; dsRNA, double-stranded; ssDNA, single-stranded HBV DNA. HBV pgRNA and total RNA levels were measured by real-time PCR. GAPDH was used as a loading control (C). HBV 3.5 kb (pgRNA), 2.4/2.1 kb (Pre-S/S RNAs) and 0.7 kb (X-RNA) RNAs were detected by Northern blot analysis (D). Data are presented as the mean ± SD from three independent experiments. **p < 0.01 compared with mock. In the mean time, CCK-8 and 3H-thymidine incorporation assays were performed to detect the potential effect of p53 on cell proliferation. Transfection of HepG2 cells with pcDNA-p53 induced only about 15% of decrease of cell proliferation 72 hr post-transfection (data not shown). The above results suggest that inhibition of HBV by p53 is not due to its interference with cell proliferation.|
|HEP_24723_sm_suppinfofig7.tif||716K||Supporting Information Fig. S7. MiR-122 expression was down-regulated by IFN-α. (A) Dose-response analysis of miR-122 induced by IFN-α. Huh7 cells were treated with the indicated concentration of IFN-α for 4h, and miR-122 expression was quantified with real-time PCR. The results were normalized to a U6 endogenous control RNU6B. The miR-122 levels in untreated cells (control) were arbitrarily set as 1.0. (B) Time course of miR-122 expression in response to IFN-α treatment. Huh7 cells were treated with 1000 U/ml of IFN-α for the indicated time, and miR-122 expression was quantified by real-time PCR. Error bars, the mean ± SD of three independent experiments. * P<0.05, ** P<0.01 compared with control.|
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